Novel glycolipid complexes and their uses

ABSTRACT

A complex of a water-soluble polymer containing hydrophobic regions and native sulfatide is disclosed. The complex may be used in assaying methods.

TECHNICAL FIELD

[0001] The present invention relates to novel forms of glycolipids andnovel methods for assaying anti-glycolipid antibodies. The preferredglycolipid is sulphatide. The novel assaying methods are particularlyadapted for the diagnosis of any disease for which elevated levels of ananti-glycolipid autoantibody is a marker. The disease may be, forinstance, neuropathy and insulin-dependent diabetes (IDDM, Type 1diabetes) including diagnoses excluding Type 2 diabetes, latecomplications associated therewith and monitoring the treatment of IDDM.The novel forms of sulphatide may potentially find use as a medicamentin the treatment of IDDM. Sulphatide, the most important antigen of thepresent invention is a glycosphingolipid having the structure:

[0002] (=galactosylceramide-3-sulphate).

[0003] The development of manifested IDDM involves an autoimmune processwhere the insulin producing cells in the islet of Langerhan in thepancreas gradually are knocked out. At the time of diagnoses 70-80% ofthe cells have been destroyed and the remaining cells will generallydisappear within a 5 year period. Prediabetic individuals are thosewhich have an ongoing destruction of insulin-producing cells but stillwithout clinical symptoms. A particular type of prediabetic individualsare non-IDDM patients whose disease will switch to IDDM.

[0004] In the context of the invention the term IDDM include manifestedIDDM as well as preforms thereof, if not otherwise specified.

[0005] Prior Art Situation

[0006] The main marker autoantibodies found in IDDM are islet cellantibodies (ICA), anti-glutamic acid decarboxylase antibodies(anti-GAD), anti-insulin antibodies (IAA), antibodies against 37 kDprotein (likely the same as tyrosine phosphatase) and anti-sulphatideantibodies. The main disadvantages of these markers are:

[0007] ICAs are determined by conventional immunohistochemistry whichmake them unsuitable for large screenings. They are normally consideredgood predictors of IDDM but their assay suffers from relatively highintra- and interassay variations.

[0008] Anti-GAD antibodies are easy to determine also in large scalescreenings, but they may not be of a strong predictive value.

[0009] IAAs are fairly easy to determine but are not present in morethan 40-50% of newly diagnosed type 1 diabetic children and is lesscommon in adult patients.

[0010] Anti-37 kD protein antibodies have a significant lower frequency(about 50%) than ICA and anti-GAD antibodies.

[0011] Both ICAs and anti-GAD antibodies have been used for thediagnoses of prediabetics.

[0012] Anti-sulohatide antibodies have been recognized as a good markerfor manifested IDDM (Buschard et al., Lancet 342 (1993) page 840-; andBuschard et al., APMIS 101 (1993) 963-970). Results supporting adiagnostic link between this type of antibodies and prediabetic forms ofIDDM have not hitherto been published. Increased titers ofanti-sulphatide antibody have been found in neuropathy patients andcould be used for identification of individuals that might developneuropathy. Part of diabetes type 2 patients also develop neuropathy. Ina preliminary study one of the present inventors has shown that diabetestype 2 patients without neuropathy did not have anti-sulphatide antibodyreactivity and there is thus a possibility that those with neuropathydevelopment will be positive and thus anti-sulphatide antibody might beuseful as a prognostic tool. See Fredman et al., J. Neurolog. 238 (1991)75-79.

[0013] Serum levels of antibodies towards glycosphingolipids have oftenbeen assayed by a combination of thin layer chromatography (TLC) of theantigens and ELISA methodology (enzyme-linked immuno sorbent assay)employing the chromatographed material as antigen for assayingoccurrence of serum antibodies (TLC-ELISA) (Fredman et al., J. Neurol.238 (1991) 75-79; and Fredman et al., J. Neurol. 240 (1993) 381-387).

[0014] The assaying methods of anti-glycolipid antibodies have beenproblematic, in particular the methods for anti-sulphatide antibodies.

[0015] Sulphated glycolipids, in particular sulphatide,lactosylceramide-3-sulphate and seminolipid and corresponding antibodieshave been suggested as diagnostic markers and therapeutic agents in thecontext of diabetes (Buschard K, WO-A-9219663).

[0016] The Main Problems the Invention Set Out to Solve

[0017] We have tried to vary the attachment of sulphatide to plasticwells, and have clear evidences that direct adsorption results in a highcoefficient of variation and often gives unspecific reactions withoutany correlation to earlier TLC-ELISA findings. The criticality ofexposing correctly both the lipid moiety and the carbohydrate moiety ofshort carbohydrate chain glycolipids has also been illustrated withmonoclonal anti sulphatide antibodies. Thus, the monoclonalanti-sulphatide antibody described by Fredman et al (Biochem. J. 251(1988) 17-22) is sensitive to changes in the lipid part. Another aspectis that galactose substituted with sulphate is a common epitope onseveral glycoproteins and glycolipids, and there are no results so farsupporting that a general reactivity towards this epitope is relevantfor the development of IDDM and its late complications. Accordingly, themain problem the invention sets out to solve relates to the presentationof glycolipid epitopes.

[0018] Another problem the invention sets out to solve is connected tothe diagnoses of prediabetic forms of IDDM.

[0019] The Objectives of the Invention

[0020] A first objective of the invention is to improve the presentationof glycolipid antigens/haptens in immunoassays and potentially also intherapy. The antigens/haptens primarily concerned exhibit shortcarbohydrate chains.

[0021] A second objective is to provide improved immunoassays formeasuring anti-glycolipid antibodies or glycolipid antigens/haptens, inparticular autoantibodies against glycosphingolipid antigens exhibitingmono- and/or disaccharide units that may be sulphated.

[0022] A third objective is to provide improved diagnostic methodsutilizing as markers auto-antibodies binding to one or more of thepreviously mentioned glycolipid antigens/haptens for diagnosing IDDMincluding preforms thereof.

[0023] A fourth objective is to provide diagnostic methods fordetermining preforms of IDDM.

[0024] The Invention

[0025] These objectives may be complied with by complexing theglycolipid antigens/haptens as defined above to a polymeric hydrophiliccarrier exhibiting hydrophobic regions. Examples of suitable carriermolecules are delipidized forms of hydrophilic proteins, such asalbumin, that are capable of associating to lipid compounds, such asfatty acids and derivatives thereof.

[0026] Accordingly a first aspect of the invention is a complex betweena water-soluble carrier polymer and a glycolipid antigen, preferably aglycosphingolipid antigen, that exhibits a mono- or disaccharide unitthat may or may not be sulphated in the 3-position of its terminalcarbohydrate unit. The water-soluble polymer is preferably a delipidizedform of a protein that have hydrophobic regions that are capable ofbinding to lipids, such as fatty acids. At the priority date the mostpreferred water-soluble polymer was delipidized albumin. Thewater-soluble polymer may be insolubilized by covalent attachment orphysical adsorption to any of the known solid phases used inimmunoassays or chromatography. Insolubilization may be carried outeither before or after complex formation with the glycolipidantigen/hapten concerned. In this aspect of the invention the best modeencompasses sulphatide as the glycolipid antigen/ hapten. The forceskeeping the glycolipid antigen/hapten complexed is believed to be mainlyhydrophobic, meaning that at the priority the preferred variant of theinventive complex is thought to be a non-covalently associated complexbetween the carrier polymer and the glycolipid.

[0027] A second aspect of the invention is an immunoassay foranti-glycolipid antibodies or glycolipid antigen/hapten utilizing theabove-mentioned glycolipid complex as an antigen. In this type ofassays, an immune complex is formed between the inventive type ofglycolipid-carrier polymer complex and the appropriate anti-glycolipidantibody (analyte) of a sample in an amount that is related, eitherqualitatively or quantitatively, to the amount of anti-glycolipidantibody in the sample. In an alternative embodiment the glycolipidantigen/hapten is the analyte which is allowed to compete with theglycolipid-carrier polymer complex as defined above for binding to adeficient amount of added anti-glycolipid antibody. In order tofacilitate measurement, further reactants that are capable of beingincorporated into the immune complex may be included, e.g. labelledreactants (labelled antibodies or labelled antigens) or insoluble orinsolubilizable reactants. Examples of labels that can be used areenzymes, enzyme substrates, cofactors, coenzymes, fluorophores, dyes,particles such as latex particles, carbon particles, metal particles,radioactive isotopes etc. The labelled reactant is used in an amount sothat the amount incorporated and/or not incorporated into the(glycolipid)—(anti-glycolipid antibody) complex becomes a measure of thelevel of analyte in the sample. In case the signal from the labelchanges upon being incorporated into the immune complex, no physicalseparation of complex-bound from non-complex-bound form of the label isnecessary before measuring the signal from the label. In case complexformation does not lead to any signal change, physical separation ofcomplex-bound from non-complex-bound form of the label becomesimperative. In case separation is accomplished one speaks aboutheterogeneous assays while otherwise the assays are called homogeneous.Physical separation of label not incorporated into the immune complexfrom label incorporated into the immune complex is normally accomplishedby utilizing a reactant that is insoluble or insolubilizable in theassay medium.

[0028] Other ways of subdividing immunoassays are in competitive andnon-competitive assays (sandwich). Still other types are agglutinationassays, turbidometric assays, precipitation assays that may or may notbe homogeneous/heterogeneous and/or competitive/non-competitive etc.

[0029] The normal conditions for immune assays are applicable, whichmeans that the pH during immune reactions normally is within the range4-11, the temperature between 0-35° C. etc. For heterogeneous assays,each of the various antigen-antibody reactions contemplated are normallyfollowed by intermediate separation and washing steps to removenon-specifically bound immune reactants and other disturbing substances.The medium for the reactions is normally water (aqueous) buffered to theappropriate pH.

[0030] The sample used derives from a body fluid and may be a bloodsample such as whole blood, serum or plasma, or any other type of samplethat may contain the anti-glycolipid antibody or glycolipidantigen/hapten concerned (urine, cerebrospinal fluid, lacrymal fluid,saliva etc.)

[0031] The immunoassays of this aspect of the invention may be usedeither for diagnoses or in order to screen for or characterizemonoclonal antibodies directed towards glycolipid antigens of theabove-mentioned type.

[0032] As per the priority date the most preferred mode of the secondaspect of the invention is given in the experimental part andencompasses delipidized albumin as the carrier polymer fixed to a solidphase and sulphatide as the glycolipid. The assay protocol encompasses athree layered sandwich assay in which the sample is incubated with thecarrier polymer which subsequently is incubated with labelled anti-humanantibody to the formation of the insolubilized ternary immune complex:glycolipid—anti-glycolipid antibody—labelled anti-antibody.

[0033] The third aspect of the invention is a method for diagnosingdisorders related to an elevated level of anti-glycolipid antigen/haptenantibody as defined above by utilizing an immunoassay of theabove-mentioned type. With respect to anti-sulphatide antibodies themethod may be applied in the context of diagnosing neuropathy or IDDMincluding prediabetic forms thereof and monotoring of treatmentregimens, such as prophylactic treatment e.g. with insulin. Usesincluded are differential diagnoses of IDDM/non-IDDM and/ordiagnoses/prediction of diabetic late complications (neuropathy,retinopathy, and neuropathy) and/or monitoring of treatment regimens ingeneral. A potential important diagnostic use included is thedetermination of preforms of IDDM in non-IDDM individuals.

[0034] A fourth aspect of the invention relates particularly to thediagnoses of prediabetic forms of IDDM utilizing elevated sample levelsof anti-sulphatide antibody as a marker for a prediabetic state in thepatient from which the sample derives. In this aspect any type of assay,particularly immunoassays, may be used, although the best results will,with the present knowledge, be obtained in case the assay is run inaccordance with the above-mentioned third aspect with anti-sulphatideantibody as the analyte.

[0035] It has been realized during the development of the presentinvention that the diagnostic use of the inventive immunoassay methodfor elevated levels of anti-sulphatide antibody may be further improvedin case it also takes into account elevated levels of other markerautoantibodies found in the context of IDDM as described above, forinstance islet cell antibodies (ICA) , anti-glutamic acid decarboxylaseantibodies (anti-GAD) anti-insulin antibodies (IAA), antibodies against37 kD protein (likely the same as tyrosine phosphatase). It is believedthat in particular a simultaneous finding of elevated levels ofanti-sulphatide antibody and anti-GAD improves the diagnostic value byincreasing the specificity.

[0036] A potent fifth aspect of the invention, is to use the complexdefined in the context of the first aspect of the invention as theactive ingredient/drug in pharmaceutical compositions. The preferredmode includes the above-mentioned novel sulphatide complexes to preventand/or delay development of diabetes (IDDM) and to the treatment ofdiseases related to late complications of diabetes (IDDM). The complexesconcerned may, for example, interact with the immunological ligandand/or immune active cells involved in the pathogenic process andthereby inhibit their binding to glycolipid/sulphatide in target cellslike α- and β-cells of the pancreas. Potentially the glycolipidcomplexes of the first aspect of the invention may also interactpharmacologically in intracellular events involved in the diseaseprocess and caused by sulphatide and/or its metabolic products. Inparticular sulphatide complexes as defined above may be used as vaccinesin order to prevent, delay or alter IDDM or its late complications.

[0037] During the priority year it has been realized that diagnoses inthe context of IDDM by assaying for anti-sulphatide antibodies may befurther improved in case one also accounts for elevated levels ofanti-GAD antibody. The improvement mainly relates to an increased in thespecificity. This type of diagnoses is not linked to any specific methodfor assaying anti-sulphatide or anti-GAD antibodies, although at thefiling date it was preferred to utilize the novel forms of glycolipidsas described for assaying anti-sulphatide antibody. Employment ofelevated levels of both anti-sulphatide and anti-GAD antibodies incombination as markers in the context of IDDM as described above is aseparate invention.

Experimental Part

[0038] Production of delipidizid albumin: Albumin was delipidized withhexane containing 5% glacial acetic acid, at +4° C. After thoroughwashing with hexane the albumin was dialyzed against Milli Q-water andlyophilized.

[0039] Biotinylated delipidized albumin: Fatty acid free albumin asdescribed above was dissolved in 0.1 M NaHCO₃ to a concentration of 10g/L. Biotin-N-hydroxy succinimide, 0.1 M in dimethyl formamide, wasadded to a final concentration of 0.02 M. The final mixture was left at20° C. for 60 min and an equal volume of phosphate buffered saline (PBS)was added. The reaction product was dialysed against 5 changes of PBS at+4° C. for 24 hs.

[0040] Preparation of sulphatide: Bovine brain was homogenized with anequal volume of water. Methanol and chloroform were then added to afinal ratio of 4:8:3 (by volume, chloroform-methanol-water). The lipidextract obtained was freed from particles by centrifugation. Lowmolecular weight components were removed by partition after addition ofchloroform and methanol to a ratio of 4:2:1 (by volume,chloroform-methanol-water). The crude lipid extract was chromatographedon silica gel. The isolated sulphatide fraction was saponified andrechromatographed on silica gel. After repeated partition the sulphatidefraction was further purified by ion-exchange chromatography. Finallythe purified fraction was dissolved in methanol and precipitated byaddition of acetone. The isolated sulphatide fraction was characterizedby mass spectrometry (FAB-MS). The recovery was about 1.5 g sulphatideper kg of bovine brain.

[0041] Adsorption of sulphatide to albumin: Sulphatide (100 nmole inchloroform-methanol-water mixture was evaporated to dryness andredissolved in 500 μl sodium acetate (0.05 M pH 4.5) and sonicated for15 min at room temperature. To this solution was added 0, 5 mLdelipidized albumin dissolved in the same buffer (2 mg/mL). The mixturewas incubated over night at room temperature under careful mixing.Thereafter the albumin with sulphatide adsorbed was precipitated by theaddition of 50 μL of 10% TCA (trichloro acetic acid) in water at +4° C.After 30 min at +4° C. the mixture was centrifuged (+4° C.) at 23,000×gfor 3 min. The pellet was suspended in 1 mL PBS and kept at +4° C. Theprocedure could also be applied to biotinylated albumin.

[0042] Binding sulphatide-albumin to a solid phase: Sulphatide-albuminwas suspended in 50 mM carbonate buffer pH 9.6 to a concentration of 2.5mmole/mL and 100 μL of this solution was added to the wells of amicrotiter plate (Nunc Storwell Maxisorp immunomoduler, Denmark) . Theplate was incubated for more than 2 hours at 37° C. and was then sealedwith parafilm and kept at +4° C. until used. Just prior to the assay,the coating solution was flicked out and the wells incubated with 100 μLof PBS containing 1% dry milk powder (w/v) for one hour in order toblock the surface for unspecific binding. The procedure could be appliedalso to sulphatide bound to biotinylated albumin.

[0043] Assay Protocol

[0044] Serum samples, 50 μL of serum diluted 1:400 with PBS containing1% milk powder were added to the wells of a microtiter plate treated asdescribed above. In case of high sulphatide titer sera, the samples werefurther diluted. The plates were then incubated at +4° C. over night. Apool of sera from blood donors (50 serum samples each individually foundto be negative for anti-sulphatide reactivity analysed with TLC-ELISA)was used as reference. A positive serum was used as an internalstandard. Each sample was analysed in triplicate. After incubation thesamples were flicked out and the wells washed with NUNC immunowasher 6times with 0.1% albumin dissolved in PBS. Thereafter 50 μL phosphataseconjugated anti-human IgG (Zymed, BioZac, Sweden) diluted 1:500 with 1%albumin in PBS were added and the plate incubated for 1 hour at roomtemperature. Unbound components was then removed by washing 5 times withPBS containing albumin (1%). Thereafter incubation was performed at +37°C. with 100 μL p-nitro phenyl phosphate (1 mg/mL) (phosphatase-substratetablets 104, Sigma, U.S.A.) dissolved in 1.0 M diethanol amine buffer pH9.8. The enzymatic reaction was stopped by adding 50 μL 3 M NaOH. Theabsorbance was the read at 405 nm.

[0045] The results on clinical samples have indicated safer diagnoses inthe context of IDDM, and have also showed, for the first time, thatelevated levels of anti-sulphatide antibody functions as a marker forpreforms of IDDM. In addition the usefulness of anti-sulphatide antibodyas a marker for monitoring treatment regimens, the results suggesteddiagnoses/prediction of diabetic late complications with this antibodyas a marker.

1. A complex between a water-soluble polymer containing hydrophobicregions and a glycolipid.
 2. The complex of claim 1, characterized inthat the polymer and the glycolipid is non-covalently bound to eachother.
 3. The complex of anyone of claims 1-2, characterized in that thewater-soluble polymer is a delipidized protein that in native form bindslipids.
 4. The complex of anyone of claims 1-3, characterized in thatthe glycolipid is a glycosphingolipid.
 5. The complex of anyone ofclaims 1-4, characterized in that the glycolipid contains one mono- ordisaccharide unit that preferably is sulphated at the 3-position in itsterminal monosaccharide unit.
 6. The complex of anyone of claims 1-5,characterized in that the glycolipid is sulphatide.
 7. The complex ofanyone of claims 1-6, characterized in that the water-soluble polymer isalbumin.
 8. The complex of anyone of claims 1-7, characterized in thatthe complex is insolubilized to a solid phase by adsorption or covalentattachment.
 9. An immunoassay of an antibody directed towards aglycolipid or of the glycolipid, characterized in that the samplecontaining the antibody or the glycolipid is contacted with a complexcontaining the glycolipid and defined in anyone of claims 1-8,optionally also with an added anti-glycolipid antibodies in a deficientamount in case the glycolipid is assayed.
 10. The immunoassay of claim9, characterized that the antibody is anti-sulphatide antibody.
 11. Theimmunoassay method of anyone of claims 9-10, characterized in that theimmunoassay is heterogeneous.
 12. The immunoassay method of anyone ofclaims 9-11, characterized in that the method is non-competitive. 13.Method of diagnoses in the context of neuropathy and IDDM, characterizedin that anti-sulphatide antibody is assayed as defined in anyone of9-11.
 14. Method of diagosing prediabetic forms of IDDM in anindividual, characterized in that a sample from the patient is assayedfor anti-sulphatide antibody and a found elevated level compared to thehealthy population is taken as a marker for a prediabetic state of thepatient.